Project Summary The rationale for the proposed research is the need for strategies to study how tumor heterogeneity and differentiation affect osteosarcoma cell differentiation, plasticity, and mechanisms of resistance to therapy. Currently, the field of cancer research suffers from a dearth of pre-clinical models that accurately recapitulate the tumor microenvironment including mechanical cues which are essential for differentiation and generation of normal cell phenotype. Therefore, the objective of the proposed research is to utilize 3D electrospun scaffolds with variable mechanical properties to leverage the effect of substrate stiffness on osteosarcoma cell phenotype, plasticity, and response to therapy. The fundamental hypothesis of this work is that when cells are cultured in 3D environments with variable mechanical properties, they will display a higher propensity for differentiation, differential activation of essential pathogenic pathways, and variable response to therapy. The proposed research will be accomplished through two specific aims. In the first specific aim, we will expand on preliminary data that suggests variable phenotype and increased plasticity in three-dimensional mechanical environments by determining the effects of the mechanical environment on the plasticity of osteosarcoma cell differentiation. Fabricated scaffolds will be assessed through scanning electron microscopy, mercury porosimetry, and uniaxial tensile testing. Osteosarcoma cells will then be cultured on scaffolds of variable mechanical properties and will be exposed to biochemical differentiation cues for induction of osteogenic, adipogenic, chondrogenic lineage commitment. Essential biochemical signaling pathways for osteosarcoma pathogenesis and markers of differentiation will be assessed via Western blot, qRT-PCR and flow cytometry. The outcomes of this specific aim will reveal the role of the mechanical environment in dictating tumor cell plasticity and phenotype. The second specific aim will elucidate how the interplay between mechanical environment and differentiation phenotype affects osteosarcoma cell response to therapy with the doxorubicin and ridaforolimus, the subject of recent clinical trials. The first two studies will serve to elucidate the molecular signatures of differentiation and essential signaling of IGF-1/mTOR and Wnt pathways that identify osteosarcoma populations as resistant or susceptible to therapy. The third study aims to validate our model by incorporating the use of primary osteosarcoma samples obtained from patients. Primary osteosarcoma cells will be treated with therapeutics as in the first two studies of this aim; the identified molecular signatures of therapeutic response will be evaluated. We aim to leverage the effects of a tunable, 3D mechanical environment to study aberrant differentiation in divergent cell populations present in osteosarcoma and how differentiation and phenotype relate to mechanisms of resistance to therapy. The proposed research has the potential to offer cancer researchers with the tools to study variable populations of tumor cells which is impossible with standard culture techniques.